5481The Scripps Research Institute Molecular Screening CenterPKA_INH_LUMI_1536_ IC50Dose-response biochemical assay for inhibitors of protein kinase A (PKA) activitySource (MLSCN Center Name): The Scripps Research Institute Molecular Screening CenterCenter Affiliation: The Scripps Research Institute, TSRIAssay Provider: Scripps FloridaNetwork: Molecular Library Screening Center Network (MLSCN)Grant Proposal Number: NAPKA is an ubiquitous serine/threonine protein kinase and belongs to the AGC kinase family. It has several functions in the cell, including regulation of immune response [1], transcription [2], cell cycle and apoptosis [3]. PKA is a cAMP dependent enzyme that exists in its native inactive form as a 4 subunit enzyme with two regulatory and two catalytic subunits. Binding of cAMP to the regulatory subunit leads to the disassembly of the complex and release of now active catalytic subunits.This screen is designed to identify inhibitors of PKA. The known PKA inhibitor Staurosporine was used as a positive control.Keywords:PKA, kinase, Protein kinase A, luciferase, luminescence, apoptosis, immune response, cAMP dependant enzyme, Scripps, titration, IC50, dose responseAssay Overview: Compounds identified from a previously described set of experiments entitled "Primary high-throughput assay for chemical inhibitors of protein kinase A (PKA) activity" (PubChem AID= 524) were selected for testing in this assay. Out of 94 compounds identified during the primary screen, all 94 compounds were assessed in dose response experiments in 10 point, 1:3 serial dilutions starting at a nominal test concentration of 60 micromolar.As with the primary screen, this assay is based on ability of PKA kinase to phosphorylate a Kemptide peptide sequence. PKA uses ATP as a donor of phosphate for the phosphorylation of the substrate, which leads to the depletion of ATP in the reaction mix. An assay kit (Kinase-Glo, Promega) was used to quantify enzyme activity. Residual amounts of ATP are measured by a secondary enzymatic reaction, during which luciferase utilizes the remaining ATP to produce luminescence. In this assay, the luminescent signal is directly proportional to the amount of ATP and inversely proportional to PKA activity. Each concentration was tested nominally in triplicate. The dose response assay was conducted in 1536 well plate format. Protocol Summary: 1.25 microliters of substrate solution containing 20 micromolar ATP and 60 micromolar Kemptide peptide (substrate) in assay buffer (50 millimolar HEPES pH 7.3, 10 millimolar MgCl2, 0.1% BSA, 2 millimolar DTT) were dispensed into a 1536 microtiter plate. 15 nanoliters of test compound, positive or negative control (50 micromolar Staurosporine and DMSO, respectively) were then added to the appropriate wells. Each compound dilution was assayed in triplicate, for a nominal total of 30 data points per dose response curve. The experiment was started by dispensing 1.25 microliters of 0.5 nanomolar PKA in assay buffer (50 millimolar HEPES pH 7.3, 10 millimolar MgCl2, 0.1% BSA, 2 millimolar DTT). After 2 hours of incubation at 25 degrees Celsius, 2.5 microliters of Kinase Glo reagent (Promega Corporation, Madison, WI) was added to each well. Plates were incubated for 10 minutes and luminescence was read on Perkin-Elmer Viewlux for 60 seconds. The percent inhibition for each well has been calculated as follows: %inhibition = (test_compound - median_positive_control)/(median_positive_control - median_negative_control)*100 where positive control is Staurosporine (Alexis Corporation, 300 nanomolar) and negative control is DMSO only. For each compound, percentage inhibitions were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for X-intercept at the 50% inhibition level of Y-intercept. In cases where the highest concentration tested (i.e. 60 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 60 micromolar. Compounds with IC50 of greater than 60 micromolar were considered inactive; compounds with IC50 of equal to or less than 60 micromolar were considered active. The activity score was calculated based on pIC50 values for compounds for which an exact IC50 value was calculated and based on the observed pIC50 range, specifically the maximum lower limit of the pIC50 value as calculated from the lowest concentration for which greater than 50% inhibition is observed. This results in a conservative estimate of the activity score for compounds for which no exact IC50 value is given while maintaining a reasonable rank order of all compounds tested.All data reported were normalized on a per-plate basis.Possible artifacts of this assay can include, but are not limited to: compounds that inhibit luciferase activity.1617879918402961281443255661888305241Activity QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration42IC50Qualified IC50 in molar: The concentration at which 50% of the inhibition is observed (relative to 100% inhibition of 300 nanomolar reference inhibitor)153LogIC50Log10 of the qualified IC50 in M concentration.12544Hill CoefficientThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper.The Hill slope has no units.12545Hill S0Y-min of the curve.12546Hill SinfY-max of the curve.12547Hill dSThe range of Y.12548Curve Chi2A measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.12549Curve R2This value indicates how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.125410Excluded PointsNumber of excluded point in the dose-response curve(counting one data point per concentration).225411Number of DatapointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as an outlier.225412Inhibition at 3.0 nMNormalized percent inhibition at 3.0 nanomolar inhibitor concentration; average of triplicate measurement.11513Inhibition at 9.1 nMNormalized percent inhibition at 9.1 nanomolar inhibitor concentration; average of triplicate measurement.11514Inhibition at 27.4 nMNormalized percent inhibition at 27.4 nanomolar inhibitor concentration; average of triplicate measurement.11515Inhibition at 82 nMNormalized percent inhibition at 82 nanomolar inhibitor concentration; average of triplicate measurement.11516Inhibition at 246 nMNormalized percent inhibition at 246 nanomolar inhibitor concentration; average of triplicate measurement.11517Inhibition at 740 nMNormalized percent inhibition at 740 nanomolar inhibitor concentration; average of triplicate measurement.11518Inhibition at 2.2 uMNormalized percent inhibition at 2.22 micromolar inhibitor concentration; average of triplicate measurement.11519Inhibition at 6.6 uMNormalized percent inhibition at 6.66 micromola inhibitor concentration; average of triplicate measurement.11520Inhibition at 20 uMNormalized percent inhibition at 20 micromolar inhibitor concentration; average of triplicate measurement.11521Inhibition at 60 uMNormalized percent inhibition at 60 micromolar inhibitor concentration; average of triplicate measurement.1153cAMP-dependent protein kinase catalytic subunit alpha isoform 1 [Homo sapiens]45060551Homo sapienshumantaxon96062184269702361=237.513-4.4341.5452.671654.283751.611836.3290.9911302152.820431918.2189.2174.8161.1150.3143.6133.6122.520074984279701201>259.643-4.221130212.92015.2199.1184.6174.4161.5152.6144.1133.2123.62007498428680161=2231.43-3.6440.835-0.268685.6785.8688-76.6590.9711302126.82013.1195.718317-0.9161.615-0.414-1.113-0.812120074984328901201>259.643-4.22113021432030.11916.6189.2177.2167.6156.7148.3137.1129.420074984524501201>259.643-4.2211302123.52015196.7185.5173.8166.8155.2144.4134.7125.720074984595402421=224.643-4.6141.0455.462676.673771.211815.9190.9911302165.32044.81927.11811.8177167.6156.1145.9135.1125.320074984622601201>259.643-4.22113021-1.620-5.519-4.518-2.317-2.4160.9150.414-4.713-0.812-0.120074984696102461>219.883-4.711302189.62051.91942.41843.81718.3166.9151.9146.4132.512220074984906801201>259.643-4.2211302120.72012.9194.5183.7172.8165.8154.1143132.5123.320074984965501201>259.643-4.2211302133.32022.11914.81810.3179.41610.5159.3146.4135.4126.120074985047001201>259.643-4.2211302117.72017.31914.71810.5175.6166.2154.4145.6135.1122.620074985064701201>259.643-4.2211302135.120341919.21810177.6169.1158.3147.81311.71210.320074985191401201>259.643-4.2211302144.22046.51936.91813.51711.3167.7155.7146135.8124.420074985291402551=211.333-4.9541.075-0.233682.89783.1238-1125.0191113021742061.91936.91818.7176.1160.1150.9143130.112-2.720074985566902961=20.8533-6.0741.2957.7256104.134796.40987.8791113021111.320108.619102.71881.31743.91621.31512.7148.413712720074985568602711=24.0893-5.3941.3257.437671.518764.08189.819111302176.52075.11959.21835.51718.3168.6156.3147.6138.1128.720074985593302631=26.6413-5.1841.1255.238688.03782.792815.729111302184.42078.519461826.61714.5167.5155.4145133.6126.120074985685902581=28.9443-5.0541.0754.578685.049780.471813.239111302180.42067.919421822.51710.6167.8155145.6135.1122.520074985715702471=217.863-4.7541.0857.2556108.6367101.381825.490.9811272053.11926.61815.2178.8167.1156.1146.5138129.420074985718902311=250.453-4.340.7651.9576111.6557109.698832.5390.9911302153.72031.51918.5189.8174.6162.1150.7143.9133.1122.220074985831901201>259.643-4.22113021212010.6191180.717116-2.7150.614-0.1131.312120074985844302581=29.3693-5.0341.2453.136670.69767.554832.290.9911302169.22064.81939.81823.2178.6160.7152146.2134.9122.520074985850901201>259.643-4.2211302127.32020.21911.6187.1172.2160.9152.2142.7131.3122.620074985851401201>259.643-4.2211302142.42045.61926.91814.2179.8166.815-0.1142.8130.6121.420074985859502641=26.2023-5.2141.156.822685.318778.496831.3390.9911302186.32072.81952.818271714.21610.1157.4146.2138.9125.420074985879901201>259.643-4.2211302139.82024.91911.1189.2175.4168.7158.1143.6134.1126.220074985948702461>219.883-4.711272190.22055.71945.71729.21615.1159.21411.9137.312620074985985302311=251.113-4.2940.8951.047672.429771.381857.990.9911302152.32034.81919.9189.2173.7162.3150.5141.9131.8121.120074986054502511=214.243-4.8541.156.627687.384780.757810.0991113021792057.91932.51817.7178.1166.5158.8146.4135.3128.320074986107002491=216.243-4.7941.1155.56668.328762.76888.979111302165.92054.11932.61817.3179.9166.7157.2144.7135.2125.420074986209302521=213.63-4.8740.5752.566157.1697154.609816.499111302181.92055.81938.51825.71712.5168.9155.4143.6133.3125.220074986222301201>259.643-4.2211302135.32035.91931.41826.81717.7169.5156.8147134.4122.920074986320501261=270.093-4.1541.1552.354654.317751.963844.5690.9911302148.62037.21918.9189.4176160.9152.2142.9133.4121.720074986469202421=224.893-4.641.4356.359655.817749.458819.390.9911302158.62045.51927.41811.3176.2164158.4147.1137.6126.520074986479902361=235.93-4.4441.5754.061657.397753.33683390.9911302155.72039.31916.7187.5173.5161.8154.3144.2133.2127.4200749371265602361=237.483-4.4342.3355.124646.807741.683861.690.98113021512044.91918.2188.5173.6166.9157143.2135123.7200749371408802361=235.983-4.4441.6356.045649.168743.123821.4790.98113021522045.11921.21813176.6166.2156.3145.7135.2125.5200749371532902301=252.293-4.2841.0452.535677.173774.638837.690.9911302152.42031.31913.8187.7173.5163.8154.814-0.1131.9123.2200749371562702531=212.583-4.941.8558.032661.96753.928811.2790.9911302167.32061.41930.31816.1177.8167.1156.7146.8138.7129.8200749371742302451=221.133-4.6840.8155.264690.546785.282822.4190.9911302167.820501928.51818.71710.6167.5156.8145.8136.8123.3200749371758202281=259.173-4.2340.7154.225673.249769.024828.7990.9811302150.32035.31924.71813.61710.2165.2156144.7134125200749424070301201>259.643-4.2211302137.72028.11915.2187.9176.8164.2155145.9134.8124.9200749424167202351=239.153-4.4141.2356.818653.402746.584819.9490.9911302154.120401926.4189.6178.9169.4158.2143.1136.9128.1200749424174801201>259.643-4.2211302123.92015.8199186.6176.2168155.6144.8134.5126.7200749424283602411=226.763-4.5741.585-0.255664.364764.6198-501.4690.9911302159.52043.91918.3183.7170.216-1.515-2.8142.7130.4120.5200749424293401201>259.643-4.2211302124.320211910.5189176.5168.1158.7149.11310.2128.3200749424422501201>259.643-4.2211302123.42016.51911.1186.4177.7167.4157.1146.9137.1125.2200749424524601201>259.643-4.2211302134.72025.71913.4187174.9162.7152.7144.6135.9126.5200749424652702361=235.613-4.4541.3953.359680.618777.259817.0290.9911272162.820341912.5186.2173.4155.1141.7133.6123.1200749424679501201>259.643-4.2211302142.720311910.7188.1173164.9154.814-0.5131.7121.4200749424789802291=254.813-4.2640.954.802665.175760.373827.2190.9811302150.92036.41920.31813.2176.6168.1154144.2135125.2200749424804601201>259.643-4.22113021282042.21924.41814.5177.3166.8155.9149.4138.81210.2200749425097202551=210.993-4.9641.356.628672.945766.318821.5690.9911302173.22064.81935.71818.31712.7168.2156.9146.4134.3125.9200749425230102581=28.8993-5.0541.7157.145662.17755.024848.990.9811302164.62068.61939.51819.9179.9169.21511.1147134.4122.7200749425455402461=220.013-4.741.1251.81673.634771.823846.4890.9911302165.52049.91928.11811.5174.4165.1153.814-1.4132121.8200749425482501201>259.643-4.2211302133.62027.91915.91810.4178.6168.5157.1143.7135.3122.9200749425497902551=211.13-4.9541.1955.846680.454774.609810.859111302178.120631937.41818.8178.6167.5156.6144.6135.1127.4200749425522202541=212.093-4.9241.357.314665.132757.818819.9190.9911302167.120611935.31819.71711169.9159.9146.8136.7123.7200749426022901201>259.643-4.2211302133.22015.81911.5189.5177.5165.9156.7145.7136.8127.8200749426034802841=21.7613-5.7541.2558.095689.216781.12186.989111302195.32094.81979.31857.317291612.61511146.9137.71210.3200749426282601201>259.643-4.22113021192013.8199.2182.417-0.116-0.315-1.7142.8131.9121200749426387202331=242.93-4.3740.7154.6496112.1987107.548815.6790.9911302156.12037.719201812.51710.8168.4154.6144.1134125.2200749426437102291=257.853-4.2441.1951.665666.647764.982865.5490.9911302150.62029.61913.7183.7173.1161.8150.6143.2132.9120.3200749426484602571=29.7793-5.0140.9754.123691.918787.796814.049111302181.520681938.518221713.1166.4154.1146.5133.1122.9200749426568602321=245.553-4.3441.1652.425660.009757.583831.9290.9911302152.92037.71920.4187.9175.4161.2151.7142.4133.4123.5200749796654902561=210.383-4.9841.7158.264664.872756.608814.3790.9911302170.82064.11937.21815.417121610.3158.5145.9137.2127.5200749796790802311=250.453-4.341.1856.025653.442747.416817.6290.9911302151.22039.21920.91812.4177.6167.9156145.8135.2125.7200749796905101201>259.643-4.2211302145.52039.91911.218-0.217116-3.415-2.6141.6130.312-0.7200749796918302331=245.043-4.3540.8651.426681.738780.3128123.8690.9711302154.32037.11917.9188.2178.4163.6150.6140.4130.3122.6200749796928502541=212.153-4.9241.1756.711675.61768.89984.989111302174.62059.51937.21818.5179.7168.1155.7147.4136.9127.4200749796955902721=23.8653-5.4141.1855.248697.852792.604811.449111302197.42091.51962.61837.41713.9169155.7146.3136.5123.6200749796966702631=26.7953-5.1741.1455.962695.964790.002815.890.9911302191.42081.41945.41825.41714168.8158.2145.8133.3124.8200749796984501201>259.643-4.2211302127.32018.41911.2185.7175.1162.2153144.3134.8123.6200749796999302421=225.673-4.5941.3354.432660.915756.483816.7890.9911302158.92046.81923.11811.7176.5166.3154.7143.1132.8124.92007497970106021001=20.6823-6.1741.1655.2286101.663796.43588.8291113021105.220106.81995.81883.21751.71625.21510.8149.3135.7124.1200749797046902541=211.523-4.9441.7459.351666.21756.859826.7690.99113021722064.919321817.51713.1169.4159.7147.9137.7128200749797061702481=217.443-4.7641.0655.929663.443757.513814.3690.9911302162.72051.719341819.9179.2167156.6146.8135.8126.1200749797067702361=237.133-4.4341.5956.537646.903740.366820.5890.9911302151.92044.91927.71812.4177166.9156.1145.7136.6128.2200749797080402351=239.543-4.440.855.238683.469778.231813.190.9911302156.32040.11921.21814.61710167.2156.5145.5135.2124.1200749797081002591=28.7053-5.0641.2854.957682.036777.079826.369111302181.32069.71941.91820.3178166.4155.6146.8135.7122200749797131502501=214.833-4.8341.4756.952662.617755.664816.9190.9911302166.12055.31932.11814.8178.8164.2155.7148138.5128.3200749797137402581=29.313-5.0341.355.086685.239780.153815.899111302182.72071.91937.41819.91710165.1153.7146.6135124200749797147202541=211.543-4.9441.1754.713680.929776.216885.0990.9811302175.92065.61932.11821.11711.216-0.6154.5148.3135.1123.8200749797182002661=25.4573-5.2640.9454.386691.508787.122811.439111302187.32074.51953.41833.81714.3169.5156.7143.8135.1125.1200749797182902551=210.923-4.9641.3758.47666.051757.58822.1190.9911302169.72063.31936.71820.31711.71611.5158.5147.6138.5126.3200749797184401281=261.793-4.2141.3455.959645.997740.038824.0390.9811282148.520471928.81816.4178.6166.9155.7146.2135.2126.320074979724070101=2357.83-3.4540.8551.628650.194748.566865.0390.9711302146.42031.81928.51814.9177.5160.4152.1142.2132.5124200749797324201201>259.643-4.2211302130.82024.91914187.8177.1167.9159.7148.1136.5126.5200749797421402421=224.623-4.6141.5656.858656.712749.854832.1890.9811302159.12046.91922.618111710169.2157.2147.1134.4124.4200749797467602411=226.543-4.5841.2555.957667.19761.233822.8690.9911302162.62043.71923.81811175.3166.9156145.5137126.6200749797495102461=219.013-4.7241.5157.363652.266744.903822.390.9811302155.72053.91929.61816.21711.9168.8157.9147.3135.1125.4200749797586802541=211.833-4.9341.1953.451679.978776.527819.890.9911302174.92063.41933.61818.3178.3163.1152.3145.4134.6121.9200749797646902371=234.433-4.4641.185-0.744664.364765.1078-178.490.9911302156.12040.81922.8186.5171.916-1.415-2.7141.7130.812-1.1200749797772901201>259.643-4.2211302129.42026.91911.8183.5172.716-0.3150141.5131.4122.7200749